Oligomerization and GTP-binding Requirements of MxA for Viral Target Recognition and Antiviral Activity against Influenza A Virus

Abstract

The IFN-induced human myxovirus resistance protein A (MxA) exhibits a broad antiviral activity against many viruses, including influenza A virus (IAV). MxA belongs to the family of dynamin-like GTPases and assembles in vitro into dimers, tetramers, and oligomeric ring-like structures. The molecular mechanism of action remains to be elucidated. Furthermore, it is not clear whether MxA exerts its antiviral activity in a monomeric and/or multimeric form. Using a set of MxA mutants that form complexes with defined stoichiometry, we observed that, in the presence of guanosine 5-O-(thiotriphosphate), purified MxA disassembled into tetramers and dimers. Dimeric forms did not further disassemble into monomers. Infection experiments revealed that besides wild-type MxA, dimeric and monomeric variants of MxA also efficiently restricted IAV at a replication step after primary transcription. Moreover, only dimeric MxA was able to form stable complexes with the nucleoprotein (NP) of IAV. MxA interacted with NP independently of other viral components. Interestingly, the dimeric form of MxA was able to efficiently bind to NP from several MxA-sensitive strains but interacted much more weakly with NP from the MxA-resistant PR8 strain derived from the H1N1 1918 lineage. Taken together, these data suggest that, during infection, a fraction of MxA disassembles into dimers that bind to NP synthesized following primary transcription in the cytoplasm, thereby preventing viral replication.The interferon system plays a pivotal role in the defense against viral infection. Interferons type I and III induce the expression of more than 300 proteins, many of them exhibiting intrinsic antiviral activity (1-3). The human myxovirus resistance protein A (MxA) 3 protein is induced exclusively by type I and type III interferons and restricts the replication of a large variety of RNA and DNA viruses (reviewed in Ref. 4). Mx proteins are members of the dynamin superfamily of large GTPases. They contain an amino-terminal globular GTPase (G) domain and a carboxy-terminal stalk domain (5, 6). The stalk domain harbors the GTPase effector domain and additional structural elements, including the L4 loop required for antiviral activity and target specificity (7-9). The G and stalk domains are connected via the bundle signaling element (BSE), which strongly resembles the BSE responsible for intramolecular signaling in dynamin (10). Mx proteins exhibit a high intrinsic rate of GTP hydrolysis, although the affinity to GTP is weak (11). Moreover, the human MxA protein forms dimers and tetramers and has the capacity to form higher oligomeric ringlike structures through a series of intra-and intermolecular interactions (5, 6, 12, 13). Introduction of mutations into the interfaces of the intermolecular interaction sites or hinge regions of MxA prevents assembly into higher-order multimeric forms. These mutations lead to the formation of MxA into tetramers, dimers, and monomers (5, 12).So far, little is known about the molecular mechanism of action of the human MxA...

Evolution and Antiviral Specificities of Interferon-Induced Mx Proteins of Bats against Ebola, Influenza, and Other RNA Viruses

“…Orthomyxoviruses might be affected by a similar mechanism. Human MxA binds the nucleoprotein of influenza A virus and THOV and prevents the translocation of viral nucleocapsids into the nucleus, the site of orthomyxovirus transcription and genome replication (29,67,68). However, incoming nucleocapsids of influenza viruses may escape the early recognition by MxA and may reach the cell nucleus to start primary transcription.…”

“…However, incoming nucleocapsids of influenza viruses may escape the early recognition by MxA and may reach the cell nucleus to start primary transcription. In this situation secondary transcription is blocked, possibly by MxA-mediated sequestration of newly synthesized viral nucleoprotein (39,67). In our future research, we will focus on detailed molecular analyses of the antiviral action of the orthologous bat Mx1 proteins, especially their action against filoviruses.…”

To Conquer the Host, Influenza Virus Is Packing It In: Interferon-Antagonistic Strategies beyond NS1

“…Among the ISG products, the Mx family of large GTPases is key to the antiviral effect of IFN against FLUAV (45,46). The human MxA protein interacts with orthomyxovirus NP, particularly if it is part of the RNPs (47)(48)(49)(50)(51). MxA acts together with the NP interactors UAP56 and URH49 (see above) (52) and possibly other IFN-induced cofactors, and it restricts the access of RNPs to the nucleus, thus impairing viral primary transcription (53).…”

Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity

“…MxA also interacts with the nucleoprotein (NP) of Thogoto virus (THOV), a tick-borne orthomyxovirus, thereby blocking the nuclear import of THOV nucleocapsids and consequently viral replication (16,17). Similar to THOV, MxA recognizes the NP of influenza A virus (FLUAV) (18,19) and appears to inhibit FLUAV replication by interfering with the transport of incoming nucleocapsids to the nucleus (20,21) and also by affecting later steps of viral replication (19,22). In the case of Vesicular stomatitis virus (VSV) nucleocapsids MxA inhibits viral transcription in the cytoplasm (23,24).…”